Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

One of the main goals of the HIV-1 vaccine field is the generation of recombinant envelope glycoprotein (Env) immunogens that can elicit protective broadly neutralizing antibody (bNAb) responses [1][2][3][4][5][6] .Such bNAbs develop in a subset of chronically infected patients and have been shown to be protective in titers achievable by vaccination [7][8][9] .Env is a class I fusion trimeric protein that engages CD4 and its CCR5 or CXCR4 coreceptor to mediate target cell fusion [10][11][12][13][14] .The env gene encodes a gp160 precursor polyprotein that is synthesized and initially trimerizes in the endoplasmic reticulum.N-linked glycosylation and furin cleavage of the gp160 protein into mature gp120 and gp41 subunits are essential for folding into its native prefusion conformation 15,16 .The Env complexes on the viral surface consist of three heterodimeric gp120-gp41 protomers.The membrane proximal external region (MPER) of gp41 is attached to the membrane by a coiled coil transmembrane domain that aids in trimerization of the complex.The gp120 subunits interact with gp41 by non-covalent interactions 17,18 , which can result in shedding of some of the gp120 and exposure of gp41 'decoys' that participate in immune evasion 19,20 .
The first-generation recombinant HIV-1 Env immunogens consisted of unstable complexes that expose non-neutralizing antibody (non-NAb) epitopes, which are normally not exposed on infectious viral Env 4,21,22 .These non-NAb epitopes are considered undesirable because they attract immunodominant responses and might distract the immune system from targeting the desired neutralizing antibody (NAb) epitopes.Furthermore, these non-native Env immunogens do not properly display several quaternary-dependent antibody epitopes [23][24][25][26] .
It took several years of iterative design to generate soluble native-like Env immunogens, including SOSIP trimers [27][28][29][30] .SOSIP modifications include the truncation of gp41 at position 664, a disulfide bond (501C-605C) to covalently link the gp120 and gp41 subunits 27 , an Ile-to-Pro mutation (I559P) to prevent conformational transitions to the post-fusion state 29 (a strategy that was also applied for many COVID-19 vaccines 31 ), and an RRRRRR (R6) multibasic motif to enhance furin cleavage 28,32 .The determination of Env SOSIP trimer structures 33,34 led to a plethora of further structure-based stabilizing mutations and novel Env trimer designs, such as single-chain (SC; also called native flexibly-linked, NFL), and uncleaved prefusion-optimized (UFO) Env trimers [35][36][37] .These SC and UFO trimers contain a flexible linker that connects the gp120 and gp41 subunits and allows a native-like conformation without the need of furin cleavage 37,38 .Several native-like Env trimers based on the SOSIP and SC designs are currently being tested in phase I clinical trials 31,39 .
Gene-based vaccines, including viral-vectored and nucleic acid immunogens (e.g., mRNA and DNA), have recently gained momentum because of their simplicity, reduced development and manufacturing costs, and advances in delivery methods [40][41][42][43] .For instance, viral vectors and mRNA-containing lipid nanoparticles (mRNA-LNPs) 43 have been shown to efficiently elicit both NAb and cellular immune responses 44,45 .Furthermore, platforms such as self-amplifying RNAs 46,47 and integrase-deficient lentivirus vectors (IDLVs) 48,49 , which allow for prolonged antigen exposure, may further enhance immune responses.Most recently, nucleic acid vaccines have gained particular importance in combatting the SARS-CoV-2 pandemic 43,50 , building an excellent safety record along the way.
During the production of Env proteins for vaccination, cells transfected with state-of-the-art HIV-1 Env designs, such as SOSIP and SC, produce the desired native-like trimers, but also monomeric, dimeric and malformed trimeric species that expose undesired epitopes (Supplementary Fig. 1).Therefore, size exclusion and/or affinity chromatography purification methods are required to obtain homogeneous protein preparations containing only native-like trimeric species 30,35,36 .As selective purification is impractical during gene-based vaccination, these Env designs may not be optimal for nucleic acid vaccination approaches.One of the strategies to alleviate this problem is the use of heterologous trimerization-inducing domains, like the GCN4 leucine zipper [51][52][53] or the bacteriophage T4 fibritin foldon 54,55 motifs, which enhance trimerization of several class I fusion proteins, including HIV-1 Env 23,[56][57][58][59] and influenza hemagglutinin (HA) [60][61][62] .However, these heterologous domains can induce aberrant Env conformations 63 , and also immunodominant antibody responses that might hinder Env-or HA-targeting NAb responses 59 .
A recombinant Env design that expresses mostly as native-like trimers would therefore be a great advance for unlocking the potential of genetic vaccination for induction of bNAbs against HIV-1.Here, we describe an Env design, named Triple Tandem Trimer (TTT), in which we genetically fused three Env protomers by flexible linkers.We generated Env TTT constructs that expressed only as trimers, the majority of which were nativelike.Using viral vector-based vaccination, we found that TTT constructs induce less non-neutralizing responses than other recombinant Env trimer designs.We also applied the TTT design for generation of influenza hemagglutinin (HA) and chimeric Env and HA constructs that express only as trimers and present native-like features.

Results
Design and characterization of a native-like Env triple tandem trimer (TTT) Ideally, an HIV-1 Env construct intended for nucleic acid-based vaccination should only express trimers and should not require furin cleavage for nativelike folding (Supplementary Fig. 1).Therefore, we conceived a triple tandem trimer (TTT) design, encompassing three successive gp140 protomers genetically linked in a single open reading frame (Fig. 1a, b).As proof of concept, we generated a BG505 SOSIP.v8.4 TTT construct.Each BG505 SOSIP.v8.4 (referred to as SOSIP.v8herein) protomer includes all of the SOSIP.v4.1 mutations 64 , as well as additional modifications (MD39) that improve thermostability and trimerization 65 .The individual mutations are detailed in the "Methods" section and Supplementary Table 1.Moreover, the R6 furin cleavage site 28 was replaced by a 15-residue flexible linker to assure furin independence (Fig. 1a, b), akin to previous uncleaved singlechain (SC) Env constructs 35,36 .Next, we linked the gp41 C-terminus of each protomer to the gp120 N-terminus of the neighboring protomer (Fig. 1a, b).The termini are separated by ~11 Å (Fig. 1b); therefore, we introduced a relatively long 11-residue flexible linker between the protomers to allow enough rotational freedom for trimer assembly.Additionally, this BG505 TTT construct contained a C-terminal StrepII-Tag for purification and immobilization purposes.To visualize how the linkers could be accommodated on the bottom of the trimer, we used AlphaFold2, which predicted that BG505 TTT could fold with a structure similar to the experimentally determined structure of a native-like BG505 Env trimer (Supplementary Fig. 2a-c), even without including PDB templates as input for the prediction (Supplementary Fig. 2d).
Furthermore, the BG505 TTT protein retained favorable antigenicity and engaged quaternary-dependent PGT145 and VRC26.25 bNAbs more efficiently than SOSIP.v8controls, while the linkers in BG505 TTT did not disrupt binding of the gp120-gp41 interface-targeting PGT151 bNAb (Fig. 1e, Supplementary Fig. 4c).The predominance of native-like species also resulted in increased presentation of epitopes targeted by nonquaternary-dependent bNAbs VRC01 and PGT121 (Fig. 1e, Supplementary Fig. 4c).Nano differential scanning fluorimetry (nanoDSF) showed that TTT displayed a midpoint melting temperature (T m ) of 76.9 °C, slightly lower than one of the most stable BG505 SOSIP proteins described to date: BG505 SOSIP.v9.3 protein (80.7 °C) 70 (Table 1, Supplementary Fig. 4b).Glycosylation plays a major role in the folding, antigenicity and immunogenicity of HIV-1 Env 71 .To interrogate the glycan shield of BG505 TTT, we performed site-specific glycan analysis and compared the glycan abundance and species at each position to those from regular furin-cleaved BG505 SOSIP.v8.This analysis revealed that the glycan occupancy and processing of BG505 TTT was comparable with that of SOSIP trimers 72 , and that key glycan bNAb epitopes are conserved across both formats (Supplementary Fig. 4d, Supplementary Table 2).
To further assess whether the TTT design allows native-like folding, we determined a crystal structure at 5.8 Å of 2G12-purified BG505 TTT in complex with PGT124 Fabs and 35O22 scFvs (Fig. 1f, Supplementary Table 3).We did not observe density for the SC and TTT linkers, probably due to their high flexibility.However, the overall conformation of BG505 TTT is similar to that of other native-like BG505 Env trimers.Furthermore, although the relatively low resolution precluded the assignment of side chains and their interactions, PGT124 and 35O22 bound BG505 TTT with similar angles of approach to those observed with other Env trimers 73 .Overall, these observations confirm that BG505 TTT folds with a conformation closely resembling those of other nativelike Env trimers.BG505 TTT elicits qualitatively improved antibody responses in mice and rabbits Simian (chimpanzee) adenovirus-derived ChAdOx1 74 and modified vaccinia Ankara (MVA) 75 are well-established vector systems for the delivery of immunogens [76][77][78][79][80][81][82][83] .ChAdOx1 was used by AstraZeneca to deliver one of the   first effective SARS-CoV-2 vaccines 50 .MVA vaccines were used for smallpox eradication and are currently being used against the 2022 mpox outbreak 79 .Since BG505 TTT immunogens may be especially suitable for gene-based vaccination, including viral vector delivery, we generated ChAdOx1 and MVA vectors encoding either BG505 SOSIP.664 or TTT.Both ChAdOx1.TTT and MVA.TTT efficiently expressed as Env trimers in transduced HeLa cells (Supplementary Fig. 6).
To determine whether the Gly-Ser TTT linkers represent a neo-epitope, we measured the binding of the sera from BG505 TTT and SOSIP immunized mice to a TTT linker-like peptide (Supplementary Fig. 9).None of the sera showed binding, indicating that TTT linkers are not immunogenic or, at least in the context of BG505 TTT, do not represent an immunodominant neo-epitope.
Next, we measured the autologous NAb titers against a BG505/T332N pseudovirus in a standard Tzm-bl assay.None of the mice showed autologous NAb activity (Supplementary Fig. 7b), consistent with earlier reports that mice do not easily generate NAbs against BG505 trimers 86 .In rabbits, despite the higher trimer-specific binding responses, BG505 TTT elicited autologous NAb responses similar to those elicited by SOSIP.664(Supplementary Fig. 8b).An explanation might be that the dominant autologous BG505 NAb epitopes are present on gp120 and BG505 gp120 can in some cases induce BG505 NAbs 22 .Furthermore, we did not detect crossneutralizing responses in the two rabbits (37194 and 37212) that developed the highest autologous NAb titers at week 42 (Supplementary Fig. 8c).
In summary, BG505 TTT efficiently elicits trimer-focused antibody responses in both mice and rabbits.In rabbits, the BG505 TTT-induced responses are able to neutralize the parental BG505 virus.
Glycans effectively mask immunodominant epitopes on BG505 TTT BG505 env lacks conserved PNGS on positions 241 and 289, which creates a large immunodominant glycan hole that attracts unwanted strain-specific Ab responses [87][88][89][90][91] .Therefore, we generated a glycan hole masked (GM) BG505 TTT construct (TTT.GM) that includes potential N-linked glycosylation sites (PNGS) at positions 241 and 289 (Supplementary Fig. 10a).The introduction of these PNGS did not affect the overall properties of the TTT.GM construct, which expressed only as trimers with conformation, thermostability and antigenicity comparable to those of the original TTT (Supplementary Fig. 10b-f, Table 1).Site-specific glycan analysis revealed that the newly incorporated N241 and N289 sites in TTT.GM were efficiently populated by oligomannose-type glycans (Supplementary Fig. 10g, Supplementary Table 7), which explained the complete abrogation of the binding of a 241/289-targeting NAb (10A 89 ) (Supplementary Fig. 10e).
All soluble Env proteins, including BG505 TTT, present a base glycan hole that constitutes an immunodominant neo-epitope and attracts a high proportion of undesired non-NAb responses [86][87][88] .In the context of TTT.GM, we attempted to cover the trimer base with glycans by adding PNGS in the TTT linkers, but were so far unsuccessful as these sites were largely unoccupied (Supplementary Fig. 10g, Supplementary Table 7) and RM19B1 and RM20A2 92 base-targeting Abs were still reactive (Supplementary Fig. 10e).The presence of the SC and TTT linkers did, however, block the epitopes of two other base-targeting Abs (RM19R, RM20G 92 ) (Supplementary Fig. 10e).
To evaluate the influence of the 241/289 glycan masking strategy on the immunogenicity of TTT constructs, we immunized rabbits with BG505 TTT.GM following a CMP schedule (Supplementary Fig. 11a).We found no significant differences in binding responses and trimer-to-gp120 ratios between the TTT and TTT.GM groups (Supplementary Fig. 11b, c, Supplementary Table 5).However, none of the rabbits vaccinated with BG505 TTT.GM developed detectable NAb titers against the parental BG505 pseudovirus (Supplementary Fig. 11d, Supplementary Table 6), consistent with the efficient masking of the immunodominant 241/289 glycan hole.Thus, we demonstrate that the TTT design can be adapted to accommodate glycans that shield some of the undesired epitopes on soluble native-like Env trimers.
The TTT design can be applied to influenza hemagglutinin Since class I viral fusion glycoproteins share a similar architecture 93 , we hypothesized that some of them, other than Env, might be amenable for the TTT design.A structural screening revealed that the location of the N-and C-termini of the influenza hemagglutinin (HA) protomers is analogous to that of HIV-1 Env.Therefore, we generated a proof-of-concept HA TTT construct encoding three NL03 (A/Netherlands/213/2003(H3N2)) protomers connected by TTT flexible linkers and with a C-terminal StrepII-Tag.The distance between the N-and C-termini of neighboring HA protomers is ~23 Å (Fig. 3b), i.e., substantially longer than the ~11 Å for HIV-1 Env (Fig. 1b).Consequently, we designed a longer 22-residue TTT linker to connect the different HA protomers (Fig. 3a, b).As controls, we also generated the corresponding HA and HA fused to a GCN4 trimerization domain (HA GCN4) (Fig. 3a).The AlphaFold2 prediction of HA TTT showed how the linkers could be accommodated on the bottom of the trimer (Supplementary Fig. 12).
The three HA constructs were expressed in HEK293F suspension cells and purified by StrepTactinXT affinity chromatography.HA TTT showed somewhat lower purification yields than HA and HA GCN4 (~3.0 mg/L versus ~7.6 mg/L and ~6.5 mg/L, respectively) (Table 1).In SEC, both HA TTT and HA GCN4 eluted as a single peak at a volume consistent with the size of HA trimers, while HA eluted as a single peak at a higher volume, indicating the presence of non-trimeric HA forms (Supplementary Fig. 13a).The bands on SDS-PAGE confirmed that HA TTT consisted of three covalently linked HA protomers (Supplementary Fig. 13b).By nsEM, purified HA TTT and HA GCN4 appeared homogeneous and contained only trimers, while HA appeared as a heterogeneous mix of monomers, dimers and some trimers (Fig. 3c).However, the size and shape of HA trimers complicate the reliable quantification of monomers, dimers and trimers.We therefore complexed the three proteins with Fabs of the bNAb CR9114 94 (Fig. 3c), which binds a conserved conformational epitope on the HA stem.nsEM-generated 2D class averages of these complexes revealed that all HA TTT and HA GCN4 particles presented three Fabs bound, as expected for trimers, while most HA particles had a single Fab bound, indicating that monomers were the predominant species.A nsEMgenerated 3D model confirmed that HA TTT-CR9114 complexes assume a conformation consistent with a previously determined high-resolution structure of a native-like H3 HA trimer in complex with CR9114 Fabs 94 (Fig. 3d).To assess the antigenicity of HA TTT, we evaluated the binding of several bNAbs, including the head-specific C05 95 and FluA-20 96 , and the stem-specific CR8020 97 , CR9114 94 , and FI6v3 98 (Fig. 3e).FI6v3 contacts a quaternary epitope located in the stem region between two neighboring protomers Thus, we considered that FI6v3 and CR9114 binding are appropriate proxies for proper folding and resistance to unwanted postfusion conversion.While the binding of CR8020, C05 and FluA-20 was similar for the three proteins, FI6v3 and CR9114 showed stronger binding to HA TTT and HA GCN4 than to the HA protein (Fig. 3e).These findings suggest that HA TTT not only forms trimers, but also trimers that are folded in the proper conformation.The nanoDSF-determined T m value of HA TTT (54.5 °C) was substantially higher than that of HA (49.0 °C), but lower than that of HA GCN4 (58.8 °C) (Supplementary Fig. 13c, Table 1).Furthermore, site-specific glycan analysis revealed that the glycosylation of HA TTT corresponds well with that of a native-like H3 HA trimer analyzed previously 99,100 (Supplementary Fig. 13d, Supplementary Table 8).
Overall, we show that the HA TTT format yields high quality and stable HA trimers, demonstrating the broad applicability of the TTT platform for the design of class I fusion proteins.

The TTT platform allows for expression of chimeric Env immunogens
The production of chimeric immunogens containing three different protomers in a single Env trimer has been previously reported 101 .However, this study relied on complex protocols involving several sequential purification methods for their isolation.We hypothesized that the TTT design would facilitate the controlled generation and isolation of chimeric immunogens.As proof of concept, we generated a chimeric Env TTT (chEnv TTT) construct encoding single-chain BG505, ConM, and AMC011 protomers connected by 11-residue TTT flexible linkers (Fig. 4a, b).We used Alpha-Fold2 to generate a predicted structure of chEnv TTT sequence for visualization purposes (Supplementary Fig. 14).Contrary to homotrimeric BG505 TTT, StreptactinXT-purified chEnv TTT contained substantial amounts of aggregates, malformed trimers, dimers and monomers, possibly due to lower compatibility between the protomers in the chEnv TTT protein (Supplementary Fig. 15).Therefore, we expressed chEnv TTT in HEK293F suspension cells and purified the resulting protein by PGT145 affinity chromatography, which selects only well-formed trimers, obtaining a yield of 0.4 mg/L (Table 1).The PGT145purified protein preparation contained 89% of covalently linked native-like trimers and no monomers or dimers (Fig. 4c, Supplementary Fig. 16a), and presented a nanoDSF-measured T m of 69.3 °C (Supplementary Fig. 16b, Table 1).This T m is lower than for BG505 TTT (76.9 °C), but comparable to those previously reported for BG505 SOSIP.v4.2 (70.7 °C) 102 , ConM SOSIP.v7(67.8 °C) 73 and AMC011.v5.2 (67.0 °C) 103 .After using SEC to obtain a homogeneous trimer preparation depleted of aggregates (Supplementary Fig. 16c), a BLI assay was performed to assess the antigenicity of chEnv TTT (Fig. 5d, Supplementary Fig. 16d).The antibody panel included bNAbs (2G12, VRC01, PGT121, PGT145, VRC026.25,PG16, PGT151) and non-NAbs (F105 and 19b), but also BG505-(43A2) 104 and ConM-specific (CM01A) 105 antibodies.PGT145-purified BG505 SOSIP.v5.2, ConM SOSIP.v7, and AMC011 SOSIP.v9trimers were included as homotrimeric controls.chEnv TTT was reactive to all bNAbs tested, and did not bind non-NAbs F105 and 19b.Except for PGT151, whose binding is affected by the presence of the SC and TTT linkers, and 2G12, which was used as a loading control, all bNAbs showed a binding signal that accounted for approximately the average of the binding signals observed for the three homotrimeric controls (Supplementary Fig. 16d), consistent with a well-folded chimeric protein.Furthermore, the 43A2 and CM01A showed reduced (approximately a third) binding to the chEnv TTT protein compared to the BG505 and ConM homotrimeric controls, respectively (Fig. 4d), indicating that only one of the protomers in chEnv TTT is reactive to these strainspecific antibodies, and thus validating its chimeric nature.nsEM-generated 3D models of the chEnv TTT in complex with 43A2 and CM01A Fabs confirmed that only one of the protomers of the chimeric protein was reactive to these strain-specific antibodies (Fig. 4e, Supplementary Fig. 16e).Site-specific glycan analysis of chEnv TTT revealed a glycosylation profile (Supplementary Fig. 16f, Supplementary Table 9) comparable to BG505 TTT (Supplementary Fig. 4d) and other native-like Env trimers.
The TTT platform allows for expression of chimeric influenza HA immunogens In the influenza field, the combination of immunogens from diverse viral strains is well established [106][107][108] .For instance, most licensed seasonal influenza vaccines are quadrivalent, i.e., contain attenuated/inactivated viral particles or recombinant HA proteins from four different strains to increase the protective coverage 109 .Furthermore, the location of the most conserved epitopes on the stem region of the HA protein has led to the design of head-chimeric proteins with head and stem regions from different viral strains 110 .The combination of several proteins with the same stalk but different head regions results in broad stalk-specific immune responses 110 .
To assess the conformation of the purified proteins, we imaged them in complex with CR9114 Fabs (Fig. 5d, h, Supplementary Table 11).nsEMgenerated 2D class averages showed that the H1 and H1/1 preparations contained mostly monomers, while the chimeric TTT and GCN4trimerized preparations contained only trimers with three CR9114 Fabs bound (Fig. 5d, h, Supplementary Table 11).Reconstructed 3D models of these complexes, using both C3 (Fig. 5d, h) and C1 symmetries (Supplementary Fig. 21), showed that the head domains of both cH125 TTT and cH125/111 TTT appeared in an open conformation, exposing the conserved trimer interface region, which has been suggested to be a target of protective Abs 114 .To prove the chimeric nature of the cH125/111 TTT protein, we imaged it in complex with Fabs of the H1 head-specific 5J8 and 2B05 115 antibodies.The resulting complexes were unstable and difficult to image.However, we were able to obtain 2D class averages in which H1 GCN4 presented three 2B05 Fabs bound, while cH125/111 TTT presented only one Fab bound, indicating that this latter protein contained only a single H1 head (Fig. 5i, Supplementary Table 11).
In summary, the TTT design can be used for the controlled generation of chimeric Env and HA TTT constructs that express only as trimers and present the expected binding of broad as well as strain-specific antibodies.

Discussion
Expression of many recombinant class I fusion glycoprotein constructs yields a heterogeneous mix of trimers, dimers and monomers, complicating their use in genetic vaccination approaches, including those based on viral vectors and mRNA.Here, we describe a novel design platform, termed 'Triple Tandem Trimer' (TTT), and validate it for the generation of HIV-1 Env and influenza HA immunogens that express only as trimers.
Class I viral glycoprotein trimers, such as HIV-1 Env, influenza HA, SARS-CoV-2 S protein and RSV F protein, generally form dimers/monomers or adopt a post-fusion conformation when their transmembrane domain is removed.Therefore, heterologous trimerization domains, such as the GCN4 leucine zipper [51][52][53] , the bacteriophage T4 fibritin foldon 54,55 and the molecular clamp 116 domains, are used to generate soluble versions of these glycoproteins that acquire stable pre-fusion trimeric conformation.However, even with such trimerization domains, not all resulting glycoprotein molecules assemble as trimers 59 .Furthermore, these trimerization domains might negatively affect the conformation 63 or antigenicity 115 of the trimerized proteins.Lastly, their presence results in the elicitation of undesirable trimerization domain-specific antibody responses 59 .For example, the clamp trimerization domain, which is based on HIV-1 gp41, was used to generate a SARS-CoV-2 spike vaccinethat elicited anti-clamp antibodies that cross-reacted with widely used HIV diagnostics when tested in a phase I trial 117 .Therefore, the development of this vaccine candidate was halted.The nature of the TTT design largely circumvents these various disadvantages and promotes the formation of stable trimers.
Protein fusion is a method widely used in the generation of recombinant proteins, as it allows for the merging of several functionalities and/or properties in a single molecule [118][119][120][121][122] .In most of these applications, the fused proteins are originally encoded by different genes and do not interact to form multimers.Moreover, the flexible linkers involved usually act as mere linkages that provide the spacing necessary for independent folding.The TTT fusion strategy here described is one of the first examples, in addition to the case of single-chain variable fragments (scFvs) 123 , in which the fused proteins are protomers of a multimeric protein and closely interact and depend on each other for proper folding and conformation.This strategy could be used in the future to assure the multimerization of any multimeric protein, provided that the N-and C-termini of neighbor protomers are relatively close to each other (like in Env and HA) or the addition of longer (and probably rigid) linkers is possible and does not affect the function or conformation of the protein.
Env and HA TTT constructs efficiently express a higher proportion of well-folded trimers than conventional designs, and therefore represent promising candidates for gene-based vaccination.In this study, we provide proof of concept for the use of Env TTT using adenovirus and poxvirus vectors.The combination with other state-of-the-art and emerging delivery technologies, such as mRNA-LNPs 43 , self-amplifying RNAs 46,47,124 and integrase-deficient lentivirus vectors (IDLVs) 48,49 , might further improve the induction of NAb responses.Here, we characterized the antigenicity of TTT proteins expressed in vitro.It would be important that future studies employing TTT constructs as nucleic acid-based vaccines evaluate relevant histological samples to confirm that TTT immunogens expressed in vivo present similar favorable antigenic profiles.
Gly-Ser linkers are generally thought to be poorly immunogenic and have been widely used in the design of fusion proteins, including FDAapproved therapeutic scFvs, such as Brolucizumab 125 and Tebentafusp 126 .
No adverse immune reactions have been associated with the Gly-Ser linkers therein.Here, we confirmed that the inserted TTT linkers do not contribute to the formation of immunodominant neo-epitopes in the context of Env.Furthermore, we attempted to shield the trimer base and TTT linkers by incorporating glycosylation motifs on the SC and TTT linkers.However, these sites were not efficiently glycosylated, probably due to the high flexibility of the linkers.Further design efforts, based on glycosylation or other strategies, will be needed to ensure the immunosilencing of the trimer base and potential neo-epitopes derived from the inclusion of stabilizing modifications, if necessary.
The combination of spike proteins from different viral strains or that target different steps of bNAb evolution is a promising approach for the generation of bNAbs against HIV-1 and influenza 4 .However, the diverse proteins are usually administered sequentially, in cocktail or as mosaic nanoparticles 107,[132][133][134] .The chimeric Env and HA TTT immunogens here described can include three different immunogens in every single trimer, which might influence the immune profile compared to other combination vaccines.Additionally, chimeric TTT proteins containing protomers to target several steps of bNAb evolution might help to simplify sequentialbased germline-targeting, shaping, and polishing vaccination strategies 135 , as well as accelerate the screening, approval, and manufacture of novel immunogens.Generating chimeric Env TTT antigens based on diverse gp140 subunits will probably require screening or additional optimization to ensure that the core residues of a chimeric TTT are compatible to improve folding and yield.
Recently, especially after the success of mRNA and viral vector COVID-19 vaccines, a growing number of studies have focused on the design of membrane-bound glycoprotein immunogens.Membrane-bound glycoprotein immunogens present several advantages compared to soluble glycoproteins, including multimerization on the membrane thereby improving their immunogenicity, lack of exposure of the immunodominant trimer base, and maintenance of epitopes that are not present in soluble glycoproteins, such as the MPER domain on HIV-1 Env [136][137][138] .We envision that TTT proteins can also be presented on the membrane of cells or the surface of nanoparticles, by selecting appropriate transmembrane domains and nanoparticle systems.Furthermore, the TTT design, whether used for soluble antigens or membrane-anchored antigens, uniquely enables the generation of chimeric immunogens.
As gene-based vaccination approaches have recently gained much momentum, methods that ensure the expression of predominantly correctly folded antigens from these platforms should facilitate capitalizing on this momentum for HIV-1, influenza, and other pathogens.The TTT platform might therefore be particularly suitable for next-generation gene-based vaccines aimed at induction of bNAbs.
The glycan masked TTT construct (TTT.GM) was obtained by introducing several modifications to the BG505 TTT construct: 1) NxT PNGS motifs in every BG505 glycosylation site, except for 137 and 156 (NxS), in order to increase the overall glycan occupancy 72 ; 2) PNGS motifs (241N, 291T) and compensatory mutations (240T, 290E) to close the BG505-specific 241/289 glycan hole that attracts narrow-NAb responses 89 ; 3) PNGS motifs by introducing G/S to N changes in the TTT linkers (GSGGSNGSGSG) to mask the immunodominant base glycan hole.
The HA, HA TTT, and HA GCN4 constructs were based on the HA sequence of the NL03 virus isolate (A/Netherlands/213/2003(H3N2), Genbank ADM31403.1),positions 8-503 (H3 numbering).Flexible 22-residue TTT linkers (GSGGSGGSGSGGSGGSGGSGSG) were used to connect the three HA protomers comprising the TTT construct.SC linkers were not required as this HA sequence does not contain protease cleavage motifs.The HA GCN4 construct was generated by fusing a GCN4-pII isoleucine zipper trimerization motif (RMKQIEDKIEEILSKIYHIENEIARIKKLIGER) 52,53,59 to the C-terminus of the HA construct via a flexible spacer linker (GSGGSGGSGGSGGS).
All constructs comprised the above-described sequences preceded by tissue plasminogen activator (MDAMKRGLCCVLLLCGAVFVSPSQEI-HARFRRGAR) or synthetic leader (MDRAKLLLLLLLLLLPQAQA) signal peptides.Untagged Env constructs presented a STOP codon after position 664.Strep-tagged constructs included an additional Twin-Strep-Tag amino acid sequence (GSGGSSAWSHPQFEKGGGSGGGSGGSAWSHPQFEKG) after positions 664 (Env) or 503 (HA), except for the GCN4-trimerized constructs, which included a Strep-TagII (GGRSGWSHPQFEK) after the GCN4 trimerization domain.D7324-tagged gp120, SOSIP.664 and SOSIP.v8proteins used for assessing antibody binding responses were expressed from constructs with a C-terminal D7324 tag (GSAPTKAKRRVVQREKR) after position 664.In every case, the underlined GS residues were encoded by a BamHI restriction site useful for cloning purposes.
Genes were codon-optimized and synthesized by Genscript or Integrated DNA Technologies (IDT), and cloned by restriction-ligation into a pPI4 plasmid.For TTT constructs, especially the ones intended to be delivered by ChAdOx1 and MVA viral vectors, we applied specific optimization considerations and manually optimized each of the three protomer units differently to avoid repeat-mediated gene instability.Subcloning into ChAdOx1 and MVA vectors was achieved by priorly introducing 5' KpnI/SmaI and 3' NotI restriction sites.
Supernatants containing proteins with C-terminal Strep-Tag peptides were purified by StrepTactinXT affinity chromatography.First, supernatants were mixed with 1 mL of BioLock biotin blocking solution (IBA, cat.no.2-0205) and 110 mL of 10x Buffer W (1 M Tris/HCl, pH 8.0 1.5 M NaCl, 10 mM EDTA) per liter of supernatant.Then, the mixtures were incubated for a minimum of 15 min at 4 °C before passing them (0.5-1.0 mL/min) through StrepTactinXT Superflow (IBA, cat.no.2-1208) resin beds packed in Econo-Column chromatography columns (Bio-Rad).Columns were washed with ten column volumes of 1x Buffer W, and elution of the retained proteins was performed with 1x BXT elution buffer (IBA, cat.no.2-1042).
The BG505 TTT protein for X-ray crystallography and the chEnv TTT protein were purified by immunoaffinity chromatography using 2G12 and PGT145 bNAbs, respectively, similarly to previously described 70 .Briefly, unpurified proteins contained in the supernatant of transfected HEK293F cells were immobilized on 2G12 or PGT145 functionalized CNBr-activated Sepharose 4B columns (Cytiva, cat.no.17-0430) by flowing at 1 mL/min or overnight incubation at 4 °C on a roller bank.After washing with three column volumes of 0.5 M NaCl and 20 mM Tris HCl pH 8.0, proteins were eluted with 3 M MgCl 2 pH 7.5.
All eluted proteins were subsequently buffer exchanged to PBS or TBS by using Vivaspin20 ultrafiltration units (Sartorius) with MWCO 50 kDa (Env) or 10 kDa (HA).Protein concentrations were calculated using A 280 values measured on a NanoDrop2000 device (Thermo Fisher Scientific) and the molecular weight and extinction coefficient values estimated by the ProtParam Expasy webtool.Some of the affinity chromatography-purified proteins were run through a Superdex200 Increase 10/300 GL column integrated in an NGC chromatography system (Bio-Rad) to obtain a profile of the content in different species (aggregates, trimers, dimers, monomers) in the protein preparations or to remove non-Env impurities from the GNL-purified proteins used in immunization experiments.A Superose6 Increase 10/300 GL column was used to remove aggregates from the PGT145-purified chEnv TTT protein preparation.

SDS-PAGE and BN-PAGE
Proteins were run through Novex Wedgewell 4-12% Tris-Glycine and NuPAGE 4-12% Bis-Tris (Thermo Fisher Scientific) polyacrylamide gels for their SDS-PAGE and BN-PAGE analyses, respectively 64 .Subsequently, gels were stained with PageBlue Protein Staining Solution (Thermo Fisher Scientific) or Colloidal Blue Staining Kit (Life Technologies), respectively.Uncropped images of the gels are presented in Supplementary Fig. 22.Proteins presented in each gel were processed in parallel.
StrepTactinXT ELISA assays were performed following the same procedures as lectin-capture assays, with the difference that StrepTactinXT coated microplates (IBA, cat.no.2-5101-001) did not require any functionalization or blocking steps prior to protein immobilization.
Biolayer interferometry (BLI) An Octet K2 (ForteBio) device was used to measure the binding of antibodies at 30 °C and 1000 rpm agitation.Purified proteins were diluted in BLI kinetics buffer (PBS/0.1% bovine serum albumin/0.02%Tween20) to 100 nM.First, test antibodies diluted in kinetics buffer were loaded on protein A sensors (ForteBio) to a final interference pattern shift of 1 nm.To obtain a baseline prior to protein association, sensors were equilibrated in kinetics buffer for 60 s.Association and dissociation of glycoproteins were measured for 300 s.Binding data were pre-processed and exported using the Octet software.
BLI experiments on cell supernatants diluted in BLI kinetics buffer were conducted similarly to described above.However, to avoid nonspecific binding to other supernatant components, we loaded antibodies to a binding threshold of 2 nm and replaced the kinetics buffer for a mock transfected supernatant for baseline and dissociation steps.When needed, the baseline step was prolonged to 300 s to allow the signal to level out.
Nano differential scanning fluorimetry (nanoDSF) Protein thermostability was evaluated with a Prometheus NT.48 instrument (NanoTemper Technologies).Proteins at a concentration of 1 mg/mL were loaded to the grade capillaries and the intrinsic fluorescence signal was measured while temperature was increased by 1 °C/min, with an excitation power of 40%.The temperatures of melting (T m ) were determined using the Prometheus NT software.

Sample preparation and analysis by LC-MS
For protein samples to be analyzed by LC-MS, three separate 50 μg aliquots were denatured for 1 h in 50 mM Tris/HCl, pH 8.0 containing 6 M of urea and 5 mM of dithiothreitol (DTT).Next, the proteins were reduced and alkylated by adding 20 mM iodoacetamide (IAA) and incubated for 1 h in the dark, followed by incubation with DTT to get rid of any residual IAA.The alkylated proteins were buffer-exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspin columns (3 kDa), and digested separately overnight using trypsin, chymotrypsin (Mass Spectrometry Grade, Promega) or alpha lytic protease (Sigma-Aldrich) at a ratio of 1:30 (w/w).The peptides were dried and extracted using C18 Zip-tip (Merck Millipore).The peptides were dried again, re-suspended in 0.1% formic acid and analyzed by nanoLC-ESI MS with an Easy-nLC 1200 (Thermo Fisher Scientific) system coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific), using stepped higher energy collision-induced dissociation (HCD) fragmentation (15, 25, 45%).Peptides were separated using an EasySpray PepMap RSLC C18 column (75 µm × 75 cm).A trapping column (PepMap 100 C18 3 μm (particle size), 75 μm × 2 cm) was used in line with the LC prior to separation with the analytical column.The LC conditions were as follows: 275 min linear gradient consisting of 0-32% acetonitrile in 0.1% formic acid over 240 min followed by 35 min of 80% acetonitrile in 0.1% formic acid.The flow rate was set to 200 nL/min.The spray voltage was set to 2.7 kV and the temperature of the heated capillary was set to 40 °C.The ion transfer tube temperature was set to 275 °C.The scan range was 400−1600 m/z.The HCD collision energy was set to 50%.Precursor and fragment detection were performed using Orbitrap at a resolution MS1 = 100,000.MS2 = 30,000.The AGC target for MS1 = 4 5 and MS2 = 5 4 and injection time: MS1 = 50 ms, MS2 = 54 ms.

Data processing of LC-MS data
Glycopeptide fragmentation data were extracted from the raw file using Byos v3.5 (Protein Metrics Inc.).The following parameters were used for data searches in Byonic: The precursor mass tolerance was set at 4 ppm and 10 ppm for fragments.Peptide modifications included in the search include: Cys carbamidomethyl, Met oxidation, Glu pyroGlu, Gln pyroGln and N deamidation.For each protease digest, a separate search node was used with digestion parameters appropriate for each protease (Trypsin RK, Chymotrypsin YFW and ALP TASV) using a semi-specific search with 2 missed cleavages.A 1% false discovery rate (FDR) was applied.All three digests were combined into a single file for downstream analysis.All charge states for a single glycopeptide were summed.The glycopeptide fragmentation data were evaluated manually for each glycopeptide; the peptide was scored as true-positive when the correct b and y fragment ions were observed along with oxonium ions corresponding to the glycan identified.The protein metrics 309 mammalian N-glycan library was modified to include sulfated glycans and phosphorylated mannose species, although no phosphorylated mannose glycans were detected on any of the samples analyzed.The relative amounts (determined by comparing the XIC of each glycopeptide, summing charge states) of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted chromatographic areas for different glycotypes with an identical peptide sequence.Glycans were categorized according to the composition detected.HexNAc(2), Hex(9−3) was classified as M9 to M3. HexNAc(3)Hex(5−6)Neu5Ac (0-4) was classified as Hybrid with HexNAc(3)Hex(5-6)Fuc( 1)NeuAc(0-1) classified as Fhybrid.These glycans constituted the high mannose category.Complex-type glycans were classified according to the number of processed antenna and fucosylation.Complex glycans are categorized as HexNAc(3) (X), HexNAc(3)(F)(X), HexNAc(4)(X), HexNAc(4)(F)(X), HexNAc(5)(X), HexNAc(5)(F)(X), HexNAc(6+)(X) and HexNAc(6+)(F)(X).
In silico protein modeling Protein molecular graphics were generated with the UCSF ChimeraX 1.4 software 145 .The AlphaFold v2.0 software (DeepMind) 146 was used to predict protein structures.
Negative-stain electron microscopy (nsEM) Purified proteins or immune complexes were imaged by nsEM essentially as described elsewhere 115,147 .Immune complexes were prepared by incubating Fabs and Env or HA proteins at greater than a 3:1 molar ratio for 2 h at room temperature.Subsequently, 3 μL of purified proteins or immune complexes (∼0.02 mg/mL) were applied onto glow discharged (20 mA for 30 s) carbon-coated 400 mesh copper grids (Electron Microscopy Sciences).After 5 s, they were negatively stained with 2% w/v uranyl formate for 60 s.Samples were imaged using a FEI Tecnai T12 electron microscope operating at 120 keV.Micrographs were collected with Leginon 148 and single particles were processed using Appion 149 , Relion 150 and XQuartz.The UCSF Chimera software was used to map the footprints and make the figures.Fabs on 2D class average and 3D model figures were colored using Photoshop.

Crystallization and data collection
To determine the structure of BG505 TTT, trimers were complexed with PGT124 Fabs and 35O22 single-chain variable fragments (scFvs) (in a 3.5:3.5:1molar ratio of Fab:scFv:trimer) at room temperature for 30 min.The resulting complexes were digested with EndoH glycosidase (New England Biolabs) at 37 °C for 30 min.Subsequently, the digested complexes were purified by size exclusion using a Superdex200 16/600 column (GE Healthcare) and concentrated to 10.7 mg/mL, before being subjected to crystallization screening at both 4 °C and 20 °C using the high-throughput CrystalMation robotic system (Rigaku) at The Scripps Research Institute (TSRI).High-quality crystals were obtained via sitting drops in 0.1 M sodium acetate, 0.2 M zinc acetate, 11.5% (w/v) PEG 3000, pH 4.83, at 4 °C.These crystals were harvested with 15% glycerol as cryoprotectant and were immediately cryo-cooled in liquid nitrogen.The diffraction data were collected at the SSRL12-1 beamlines (Supplementary Table 3).

Fig. 1 |
Fig. 1 | Design and biophysical characterization of a triple tandem trimer Env construct.a Linear representation of SOSIP, single-chain (SC) and triple tandem trimer (TTT) Env constructs.The SOSIP construct presents a R6 furin cleavage motif (black triangle) between gp120 and gp140 subunits, which is replaced by SC linkers (green lines) in SC and TTT constructs.The TTT construct is composed of three SC protomers fused by TTT linkers (purple discontinuous lines).N-and C-termini of each protomer are colored in yellow and red, respectively.b Expected position of TTT linkers on the structure of a BG505.SOSIP trimer (PDB 5CEZ).The distance, in angstrom (Å), between the C ɑ atoms of N-(Glu32, HxB2 numbering) and C-termini (Asp664) of neighbor protomers is indicated.c SEC profiles of StrepTactinXT-purified BG505 SOSIP.v8,SOSIP.v8+ furin, SC and TTT protein preparations on a Superdex 200 Increase 10/300 GL column.d 2D class averages generated by nsEM analysis of the StrepTactinXT-purified SOSIP.v8,SC and TTT protein preparations.Percentages of native-like trimers (green), malformed trimers (yellow) and monomers/dimers (red) are indicated.2D class averages representing non-native-like particles are shaded in the corresponding yellow or red colors.e StrepTactinXT ELISA assay with StrepTactinXT-purified SOSIP.v8,SC and TTT protein preparations against a panel of bNAbs (2G12, VRC01, PGT121, PGT145, VRC26.25,PGT151) and non-NAbs (F105).Heatmap values correspond to the ratio between the areas under the curve (AUC) of each Ab and the one of 2G12, calculated using the binding curves in Supplementary Fig. 4c.f Crystal structure of 2G12-purified BG505 TTT (blue) in complex with PGT124 Fabs (dark pink) and 35O22 scFvs (dark yellow) at 5.8 Å resolution.

Fig. 3 |
Fig. 3 | Design and biophysical characterization of a triple tandem trimer HA construct.a Linear representation of standard (HA), GCN4-trimerized (HA GCN4), and triple tandem trimer (HA TTT) HA constructs.The TTT construct is composed of three HA protomers fused by TTT linkers (discontinuous purple lines).The N-and C-termini of each protomer are colored in yellow and red, respectively.b Expected position of TTT linkers on the structure of a HK05 trimer (A/Hong Kong/4443/2005(H3N2), PDB 2YP7).The distance, in angstrom (Å), between the C ɑ atoms of N-(Asn8, PDB numbering) and C-termini (Lys503) of neighbor protomers are indicated.c 2D class averages generated by nsEM analysis of the StrepTactinXT-purified H3 HA, HA GCN4, and HA TTT protein preparations, both unliganded and in complex with a CR9114 Fab (+ CR9114 Fab).d nsEMgenerated 3D model of the purified HA TTT protein in complex with CR9114 Fab, independently (up) and superimposed with a previously determined structure of a similar HK68 trimer (A/Hong Kong/1/1968(H3N2)) + CR9114 complex (PDB 4FQY) (down).e StrepTactinXT ELISA with StrepTactinXT-purified HA, HA GCN4, and HA TTT protein preparations against a panel of head-(C05, FluA-20) and stem-specific (CR8020, CR9114, FI6v3) antibodies.Dots and error bars represent the average and standard deviation of values obtained in two independent experiments.

Fig. 4 |
Fig. 4 | Design and biophysical characterization of a chimeric Env TTT (chEnv TTT) construct.a Linear representation of the chEnv TTT construct, composed of single-chain BG505, ConM, and AMC011 protomers connected by TTT linkers (purple discontinuous lines).N-and C-termini of each protomer are colored in yellow and red, respectively.b Position of TTT linkers on the AlphaFold2-predicted structure of chEnv TTT (Supplementary Fig. 14).c nsEM-generated 2D class averages of the PGT145-purified chEnv TTT protein.Percentages of native-like trimers (green), malformed trimers (yellow), and monomers/dimers (red) are indicated.2D class averages representing non-native-like particles are shaded in the corresponding yellow or red colors.d ProtA BLI assay with PGT145 + SEC-purified chEnv TTT protein and BG505-(43A2) and ConM-specific (CM01A) antibodies.PGT145-purified BG505 SOSIP.v5.2, ConM SOSIP.v7 and AMC011 SOSIP.v9 were included as homotrimeric controls.The experiment was performed in duplicate, and the curves shown correspond to one of these repetitions.e nsEM-generated 3D models of the chEnv TTT protein in complex with 43A2 and CM01A Fabs.

Table 1 |
Production yields and biophysical characterization of the different Env and HA proteins 1urification method:1StreptactinXT, 2 GNL, 3 PGT145, 4 PGT145 + SEC.A SEC step was performed only when explicitly indicated.